The overall goal of the proposed project is to develop a robust and relatively low-cost device that can perform analytical micro-scale titrations on precious NMR samples using pre-existing NMR probes and hardware. Liquid-state, high resolution NMR spectroscopy has proven to be an extremely valuable tool in the biomedical sciences, with applications including the structural and biophysical characterization of biologic macromolecules, physicochemical characterization of small molecules, and the analysis of complex biologic specimens such as human serum, urine and homogenized tissue extracts. A widely utilized experimental protocol involves the collection of successive NMR spectra on a biological specimen during a stepwise titration of a secondary chemical or biological component. Common examples include titration of acid or base in a pH titration to measure site-specific pKa values in macromolecules, titration of a labeled biomolecule (e.g. protein) with a small molecule or another macromolecule for biophysical characterization of ligand-binding, or titrations of chemical mixtures during screening of therapeutic targets for drug development. However, current methodologies for performing these NMR titrations are either prohibitively costly for most academic settings or inefficient in terms of experimental effort or the consumption of often highly precious NMR sample material. In response, we are proposing to develop a low-cost device for automating NMR titrations using a single NMR sample, which could be adapted to any existing, commercial high-resolution NMR spectrometer. Our design fits two motor-driven, non-magnetic syringes to the edges of a standard NMR tube holder (currently used to insert the sample into central bore of the high field magnet), and will include two lengths of small diameter tubing connected to their respective syringes. In a novel application, non-magnetic Piezo-electric motorswill control the syringes, one containing a concentrated solution of titrant and the other dedicated to sample mixing. The availability of such a device would be highly valuable to a wide diversity of biomedical researchers and would have applications in the investigation of essentially all human diseases.

Public Health Relevance

High-resolution NMR spectroscopy is a widely utilized experimental tool in biomedical research. One of its primary uses is the investigation of biological macromolecules involved in the development of human diseases. NMR spectroscopy describes the three-dimensional structures of these macromolecules in solution, as well as, their dynamic behavior. NMR spectroscopy is also used in the analysis of human bodily fluids or homogenized tissue extracts. Such studies help identify metabolic changes associated with many diseases, contributing both to the development of diagnostic tests and to better understand the underlying disease mechanism. In all of the above applications, researchers often desire to understand the response of a target molecule to increasing concentrations of a second molecule. Known as a titration, these sorts of experiment can help in the identification of specific components in complex mixtures, define how two molecules interact, and serve a critical role in the development of new therapeutic agents. Current instrumentation and methodology for the performance of such NMR titrations are particularly troublesome, wasting significant time and resources. We are proposing to build a device that will automate such NMR titrations, without wasting any of the precious specimen being analyzed, and will be adaptable to any of the current NMR instrumentation. The device is designed to be simple and robust and should greatly enhance the pace of these critical experiments, which have implications for nearly all areas of current biomedical research.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRR1-BT-7 (01))
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Friedman, Fred K
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Yale University
Schools of Medicine
New Haven
United States
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